The binding of DYNLL2 to myosin Va requires alternatively spliced exon B and stabilizes a portion of the myosin's coiled-coil domain.

The myosin Va light chain DYNLL2 has been proposed to function as an adaptor to link the myosin to certain cargo. Here, we mapped the binding site for DYNLL2 within the myosin Va heavy chain. Copurification and pull-down experiments showed that the heavy chain contains a single DYNLL2 binding site and that this site resides within a discontinuity in the myosin's central coiled-coil domain. Importantly, exon B, an alternatively spliced, three-amino acid exon, is a part of this binding site, and we show in the context of full-length myosin Va that this exon is required for DYNLL2-myosin Va interaction. We investigated the effect of DYNLL2 binding on the structure of a myosin Va heavy chain fragment that contains the DYNLL2 binding site and flanking sequence, only parts of which are strongly predicted to form a coiled coil. Circular dichroism measurements revealed a DYNLL2-induced change in the secondary structure of this dimeric myosin fragment that is consistent with an increase in alpha-helical coiled-coil content. Moreover, the binding of DYNLL2 considerably stabilizes this heavy chain fragment against thermal denaturation. Analytical ultracentrifugation yielded an apparent association constant of approximately 3 x 10(6) M(-1) for the interaction of DYNLL2 with the dimeric myosin fragment. Together, these data show that alternative splicing of the myosin Va heavy chain controls DYNLL2-myosin Va interaction and that DYNLL2 binding alters the structure of a portion of the myosin's coiled-coil domain. These results suggest that exon B could have a significant impact on the conformation and regulatory folding of native myosin Va, as well as on its interaction with certain cargos.